Phosphoinositides are key lipid second messengers in cellular signaling, with phosphatidylinositol (PI) dependent signaling pathways playing central roles in the regulation of many cellular processes. Disruption of these pathways is common to many disease states, including inflammation, diabetes, cardiovascular disease, and cancer. Because the activity of PI second messengers is determined by their phosphorylation state, the enzymes that act to modify these lipids are central to the correct execution of PI dependent signaling pathways.
In particular, phosphatidylinositol 3-kinase (PI 3-K) is important in pathways mediating cell proliferation, survival, differentiation and motility. Inhibitors of PI 3-K have been used to confirm the cellular functions of PI 3-K, but thus far, such inhibitors have not been deemed suitable for therapeutic uses because of problems such as toxicity and low selectivity. The PI 3-K family of heterodimeric lipid kinases is known primarily for its involvement in the phosphorylation of inositol lipids via transfer of the γ phosphate of ATP to the D-3 position of the inositol ring of PI, PI(4)P, and PI(4,5)P2 giving rise to PI(3)P, PI(3,4)P2, and PI(3,4,5)P3 respectively. FIG. 1 shows an overview of these phosphoinositide metabolic pathways. PI(4,5)P2 is a minor component of the plasma membrane's inner leaflet, and is part of a second messenger system that transduces many hormone signals. When not effected by PI 3-K, the PI(4,5)P2 pathway includes a receptor with seven transmembrane segments, a heterotrimeric G-protein, and a specific protein kinase phopholipase C (PLC). Ligand binding to the receptor activates the G protein, Gq, whose membrane-anchored α subunit in complex with GTP diffuses laterally along the plasma membrane to activate the membrane-bound PLC. As shown in FIG. 1, the activated PLC catalyzes the hydrolysis of PI(4,5)P2 at its glycero-phospho bond, yielding inositol-1,4,5-trisphosphate (Ins(1,4,5)P3 and diacylglycerol (DG).
PI 3-kinase can be activated by tyrosine kinase receptors in response to growth factor stimulation. As discussed above, PI 3-kinase is then involved in catalyzing the formation of PI(3,4,5)P3 via phosphorylation of its substrate (PI(4,5)P2. By increasing cellular levels of PI(3,4,5)P3, PI 3-K induces the formation of defined molecular complexes that act in signal transduction pathways. Notably, PI 3-K activity suppresses apoptosis and promotes cell survival through activation of its downstream target, PKB/Akt. PI(3,4,5)P3 signaling is regulated by its formation and by its conversion into PI(4,5)P2. The lipid phosphatases PTEN and SHIP are two enzymes that both act to decrease the cellular levels of PI(3,4,5)P3 by conversion either to PI(4,5)P2 or PI(3,4)P2.
There is considerable evidence that the activity of PI 3-K and the regulation of the level of its lipid products, in particular PI(3,4,5)P3, is often defective in tumorigenesis, as reported in D. Roymans et al., Phosphatidylinositol 3-kinases in Tumor Progression, 268 Eur. J. Biochem. 487 (2001). PI 3-K activity and elevated PI(3,4,5)P3 levels appear to contribute to cancer progression via constitutive activation of PKB/Akt, as reported in T. Franke et al., PI3K: Downstream AKTion Blocks Apoptosis, 88 Cell 437 (1997). Activated PKB/Akt provides a cell survival signal that blocks apoptosis and promotes survival following growth factor withdrawal or detachment from the cellular matrix. W. Phillips et al., Increased Levels of Phosphatidylinositol 3-kinase Activity in Colorectal Tumors, 83 Cancer 41 (1998) establish findings that elevated PI 3-K levels have been observed in some cancers. Further, experiments have indicated that cellular transformation is PI 3-K dependent. D. Roymans et al., 268 Eur. J. Biochem. 487, A. Klippel et al., Activation of Phosphatidy linositol 3-kinase is Sufficient for Cell Cycle Entry and Promotes Cellular Changes Characteristic of Oncogenic Transformation, 18 Mol. Cell Biol. 5699 (1998). The gene encoding the catalytic subunit of PI 3-K, PIK3CA, is an oncogene which is amplified in ovarian and cervical cancers. In addition, mutations that affect the regulation of PI 3-K signaling also contribute to tumorigenesis. PTEN is a tumor suppressor that is deleted or mutated in many cancer types. By converting PI(3,4,5)P3 to PI(4,5)P2, PTEN acts as a negative regulator of PKB/Akt activation by PI 3-K. Loss of PTEN activity results in abnormal activation of PKB/Akt and suppression of apoptosis. The lipid phosphatase SHIP also acts as a negative regulator PKB/Akt activity. Ablation of SHIP in transgenic mice leads to chronic hyperplasia, and loss of SHIP activity is one characteristic of chronic myelogenous leukemia, providing additional evidence linking the loss of regulation of PI(3,4,5)P3 levels with an abnormal proliferative state.
From the above discussion, it is clear that there is a need in the art for assaying methods and assay kits for measurement of either PI 3-K activity or presence of phosphoinositide products of PI 3-K activity in tissues, as such have the potential to become powerful molecular diagnostic tools. In addition, it is clear that there is a need in the art for assay platforms that are developed for measurement of PI3-K activity in clinical samples, and for use in vitro assaying methods for novel PI 3-K inhibitors.